This project addresses fundamental questions in developmental biology using the developing leg of Drosophila melanogaster as the model system. In addition to having antero-posterior (AP) and dorso- ventral (DV) axes, the mature leg, as all appendages, also has a proximo-distal (PD) axis. Unlike the AP and DV axes, the PD axis must form de novo, within each thoracic hemisegment of a developing embryo. In the fly, the leg primordia are first identifiable as a cluster of ~30 cells that express the homeodomain-encoding Distalless (Dll) gene. By the end of larval development, the presumptive leg has ~20,000 cells and several defined domains along the PD axis. The goal of this project is understand how these cells obtain their positional information. Generally, a 'bottom-up' approach is used, in which cis-regulatory modules (CRMs) that drive the expression of PD genes are dissected in detail, to identify the upstream inputs that control their activities. In ths way, a network of CRM and gene activities will be generated. In the next funding period, particular attention will be given to 1) the most proximal PD domain, which co-expresses homothorax (hth), which encodes a homeodomain transcription factor, and teashirt (tsh), which encodes a Zn-finger nuclear factor and 2) the most distal PD domain, which is further elaborated due to the activities of the Epidermal Growth Factor Receptor pathway. In addition, the role of two 'ventral-identity' factors, encoded by the Drosophila homologs of vertebrate Sp8, dSp1 and buttonhead (btd), will be investigated, using both a CRM-centric and target gene approaches. Altogether, these experiments will lead to a better understanding of how positional information is generated in dividing cells, using transcription factors and signaling pathways that are conserved from flies to man.